The present invention relates to a process for the production of a mammalian gelatinase catalytic domain protein, to a synthetic gene expressing the gelatinase catalytic domain, to a purified mammalian gelatinase catalytic domain protein, to pharmaceutical compositions which include the mammalian gelatinase catalytic domain protein and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment.
Gelatinases participate in connective tissue remodeling (Woessner J. F., FASEB J., 1991;5:2145-2154) by degrading protein substrates like denatured collagens (gelatins), Type IV collagen (Morel F., et al., Biochem. Biophys. Res. Commun., 1993;191:269-274), and elastin (Senior R. M., et al., J. Biol. Chem., 1991;266:7870-7875). Together with other members in the matrix metalloproteinase family, gelatinases have been implicated in several pathological processes involving connective tissue degradation, such as cancer cell invasion and metastasis (Stetler-Stevenson W., et al., FASEB J., 1993;7:1434-1441).
The potential utility of matrix metalloproteinase inhibitors in controlling these pathological processes has stimulated structure-function studies for this family of zinc-containing enzymes. All matrix metalloproteinases have a signal peptide for secretion, a propeptide containing a cysteine as zinc ligand for maintaining enzyme latency (Van Wart H. E., et al., Proc. Natl. Acad. Sci. USA, 1990;87:5578-5582), and a catalytic domain containing three histidines as zinc ligands for enzymatic activity. Collagenases, stromelysins, and gelatinases have a hemopexin-like C-terminal domain, while matrilysin lacks such domain. Studies with C-terminally truncated stromelysin (Ye Q. -Z., et al., Biochemistry, 1992;31:11231-11235; Marcy A. I., et al., Biochemistry, 1991;30:6476-6483; copending U.S. patent application Ser. No. 08/281,313, filed Jul. 27, 1994, which is a continuation application of U.S. patent application Ser. No. 08/012,705 filed Feb. 3, 1993, discloses a process for the production of the catalytic domain, without propeptide, of human stromelysin), collagenase (Schnierer S., et al., Biochem. Biophys. Res. Commun., 1993;191:319-326) and gelatinase (Murphy G., et al., Biochem. J., 1992;283:637-641) have shown that the catalytic domain is sufficient for peptidase and proteinase activity. However, the C-terminal domain is required for collagenase to degrade helical collagens (Schnierer S., et al., Biochem. Biophys. Res. Commun., 1993;191:319-326; Sanchez-Lopez R., et al., J. Biol. Chem., 1993;268:7238-7247; Hirose T., et al., Proc. Natl. Acad. Sci. USA, 1993;90:2569-2573), and it is involved in binding with TIMPs (Murphy G., et al., Biochem. J., 1992;283:637-641; Baragi V., et al., J. Biol. Chem., 1994;269:12692-12697; Fridman R., et al., J. Biol. Chem., 1992;267:15398-15405) Both 72 kDa and 92 kDa gelatinases have an insert (about 174 amino acid residues) at the catalytic domain, which is homologous to the Type II domain in fibronectin and has the ability to bind gelatin (Collier I. E., et al., J. Biol. Chem., 1992;267:6776-6781; Banyai L., et al., Biochem. J., 1994;298:403-407). The 92 kDa gelatinase has an extra collagen-like insert between the catalytic domain and the C-terminal domain.
The C-terminally truncated gelatinase generated by Murphy et al. (Murphy G., et al., Biochem. J., 1992;283:637-641) contained the 19 kDa fibronectin-like insert and had activity similar to the full length gelatinase against peptide and protein substrates. Recently, Murphy, et al., (Murphy G., et al., J. Biol. Chem., 1994;269:6632-6636) also constructed a mutant of the full length 72 kDa gelatinase with deletion of the fibronectin-like insert, and the deletion mutant had a similar activity in degrading a fluorogenic peptide but a reduced activity against gelatin and casein. With deletion of the fibronectin-like insert, the catalytic domain of gelatinase is homologous to that of other matrix metalloproteinases (Murphy G. J. P., et al., FEBS Lett., 1991;289:4-7). We reconstructed the catalytic domain of human 72 kDa gelatinase (GCD) by connecting the two peptide fragments for the catalytic domain with deletion of the insert (Seq ID No: 1) (FIG. 1). The present GCD lacks both the C-terminal hemopexin-like domain and the fibronectin-like insert. We demonstrate here that the reconstructed 19 kDa GCD expressed in E. coli from a synthetic gene is a competent peptidase and proteinase.
The object of the present invention is the expression, purification, and characterization of a 19 kDa gelatinase catalytic domain (GCD) protein lacking the propeptide, the C-terminal fragment and the fibronectin-like insert (Seq ID No: 1) (FIG. 1). The removal of the propeptide eliminates the need for proteolytic or chemical activation, the removal of the C-terminal fragment removes autolytic sites, thereby making the protein resistant to autodegradation, and the removal of the fibronectin insert (19 kDa) reduces the protein size of the catalytic domain to 19 kDa. The active and stable protein with a mass of 19 kDa is suitable for structure determination by nuclear magnetic resonance spectroscopy and X-ray crystallography, as well as mechanistic studies of catalysis and inhibition. Additionally, the protein is useful in the therapy of various disease states.